Variant classification summary

NM_000321.2:c.1027_1028del

RB1 · NP_000312.2:p.(Leu343SerfsTer3) · NM_000321.2

GRCh37: chr13:48941714 ACT>A · GRCh38: chr13:48367578 ACT>A

Gene: RB1 Transcript: NM_000321.2

Final call

Pathogenic

PVS1 very strong PM1 moderate PM2 supporting

Variant details

Gene

RB1

Transcript

NM_000321.2

Protein

NP_000312.2:p.(Leu343SerfsTer3)

gnomAD AF

ClinVar

Pathogenic

OncoKB

Likely Oncogenic

Classification rationale

Interpretation summary

Generated evidence synthesis

1

NM_000321.2:c.1027_1028del (p.Leu343SerfsTer3) is a frameshift deletion in exon 10 of the RB1 gene, predicted to cause nonsense-mediated decay and complete loss of protein function. RB1 is a well-established tumor suppressor where loss of function is the accepted disease mechanism for retinoblastoma.

2

This variant is absent from gnomAD v2.1, v4.1, and gnomAD-Canada, consistent with a rare pathogenic variant not observed in the general population.

3

The frameshift at codon 343 removes the entire RB1 pocket domain, a critical functional domain for E2F binding and tumor suppressor activity.

4

ClinVar classifies this variant as Pathogenic (Variation ID: 3236941, 1-star, single submitter). No variant-specific functional studies or clinical case reports were identified in the peer-reviewed literature.

Final determination: Generic ACMG/AMP 2015 fallback rules support a Pathogenic classification based on the observed combination of very strong, strong, moderate, and supporting pathogenic criteria.

Criteria assessment

ACMG/AMP criteria review

Criteria shown when status is available

All criteria require review: For research and educational purposes only.

Criterion	Status	Rationale	Evidence used
PVS1	Met	NM_000321.2:c.1027_1028del is a frameshift deletion in exon 10 of 27, predicted to introduce a premature termination codon at p.(Leu343SerfsTer3). NMD is expected as the stop codon occurs well before the last exon-exon junction. RB1 is a well-established tumor suppressor gene where loss of function is the accepted disease mechanism for retinoblastoma and associated cancers. Under ClinGen SVI PVS1 recommendations (PMC6185798), frameshift null variants in genes with established LOF mechanism are assigned PVS1 at very strong strength.	pvs1_generic_framework pvs1_gene_context pvs1_variant_assessment
PS1	N/A	PS1 applies when the same amino acid change has been established as pathogenic via a different nucleotide change. This is a frameshift deletion introducing a premature termination codon, not a nucleotide substitution producing the same amino acid change.
PS2	Not assessed	No de novo data available for this variant. No published studies or clinical reports documenting de novo occurrence were identified in the literature reviewed.
PS3	Not met	No functional studies directly testing NM_000321.2:c.1027_1028del were identified. Seven full-text publications were reviewed; none contained variant-specific functional data. OncoKB classifies this variant as Likely Oncogenic with Likely Loss-of-function based on curated literature context, but the underlying publications do not test this specific variant experimentally.	oncokb
PS4	Not met	ClinVar submission SCV005046057 reports 2 cases (1 bilateral, 1 unilateral) from 1 pedigree, but this is a single submitter with no validated publication trail. The PMIDs cited by the ClinVar entry are all general practice guidelines (e.g., ACMG SF lists, NSGC counseling recommendations), not clinical studies reporting case-level data for this variant. No peer-reviewed publication documenting affected individuals with this variant was identified in the literature reviewed.	clinvar
PS5	N/A	Skipped per directive; not assessed.
PM1	Met	This frameshift variant truncates the RB1 protein at p.Leu343, completely removing the pocket domain (spanning approximately residues 380-785), which is the critical functional domain responsible for E2F binding and tumor suppressor activity. The RB1 pocket domain is one of the most well-characterized functional domains in tumor biology. Although the variant is not located within a statistically significant hotspot at cancerhotspots.org, it removes a well-established critical functional domain, satisfying PM1 at moderate strength per domain-level application rules.	pvs1_gene_context
PM2	Met	This variant is absent from gnomAD v2.1 (exomes), gnomAD v4.1 (exomes), and gnomAD-Canada v1.0 (genomes), supporting a pathogenic role as a rare variant absent from the general population.	gnomad_v2 gnomad_v4 gnomad_canada
PM3	N/A	Skipped per directive.
PM4	N/A	PM4 applies to in-frame deletions/insertions or stop-loss variants that change protein length without a null effect. This variant is a frameshift deletion predicted to cause NMD and complete loss of function, which is already captured by PVS1. PM4 is not applied in addition to PVS1 for the same variant under ACMG/AMP rules to avoid double-counting.
PM5	N/A	PM5 applies when a different missense change at the same residue has been established as pathogenic. This is a frameshift deletion, not a missense variant. The PM5 candidate search confirmed no classic same-residue PM5 semantics are parseable from a frameshift variant.
PM6	Not assessed	No de novo data available for this variant. No published reports documenting de novo occurrence were identified.
PP1	Not met	The ClinVar submission SCV005046057 extracted a PP1 signal (2 cases in 1 pedigree: 1 bilateral, 1 unilateral), but this is from a single submitter with no validated publication trail. The evidence is insufficient to apply PP1 at any strength level in the absence of an independently verifiable published segregation study.	clinvar
PP2	N/A	PP2 applies to missense variants in genes where missense variants are a common disease mechanism and benign missense variation is rare. This is a frameshift deletion, not a missense variant. Additionally, RB1 disease is primarily driven by truncating LOF variants, making PP2 inapplicable.
PP3	Not met	REVEL and BayesDel scores are not available for this variant as it is a deletion, not a single nucleotide variant. SpliceAI predicts no significant splice impact (max delta score = 0.05). No computational evidence supports a damaging effect.	spliceai
PP4	Not assessed	Patient phenotype data is not available in the case materials. PP4 requires that the patient's phenotype or family history is highly specific for the disease. Without access to the proband's clinical presentation, this criterion cannot be assessed.
PP5	Not met	This variant is classified as Pathogenic in ClinVar (Variation ID: 3236941) with review status 'criteria provided, single submitter' (1-star). Under the applicable PP5 rule, only ClinVar entries with 3-star expert panel review status qualify for PP5 at supporting strength. The single submitter classification does not meet this threshold.	clinvar
BA1	Not met	This variant is absent from gnomAD v2.1, v4.1, and gnomAD-Canada. The allele frequency is 0.0%, which is well below the >1% BA1 threshold.	gnomad_v2 gnomad_v4 gnomad_canada
BS1	Not met	This variant is absent from all gnomAD populations. The allele frequency is 0.0%, which is below the BS1 threshold of >0.3% for non-VCEP application.	gnomad_v2 gnomad_v4 gnomad_canada
BS2	Not assessed	No data available regarding observation of this variant in healthy adult controls. The variant is absent from gnomAD, which precludes assessment of BS2.
BS3	Not met	No functional studies demonstrating a neutral or benign effect of this variant were identified. All reviewed publications address RB1 function at the gene level without testing NM_000321.2:c.1027_1028del specifically.
BS4	Not assessed	No segregation data is available for this variant. BS4 requires evidence that the variant does not segregate with disease in affected family members.
BP1	N/A	BP1 applies to missense variants in genes where primarily truncating variants cause disease. This is a frameshift truncating variant in RB1, where truncating LOF variants are the established disease mechanism. BP1 is inapplicable to truncating variants.
BP2	Not assessed	No data available regarding observation of this variant in trans with a known pathogenic variant. BP2 cannot be assessed without such evidence.
BP3	N/A	BP3 applies to in-frame deletions or insertions in repetitive regions without a known function. This is a frameshift deletion, not an in-frame variant.
BP4	Not met	REVEL and BayesDel are not available (not SNV). SpliceAI predicts no significant splice impact (max delta 0.05). No computational evidence supports a benign effect, and no multiple lines of benign computational evidence exist.	spliceai
BP5	Not assessed	No data available regarding observation of this variant in a case with an alternate molecular basis for disease.
BP6	Not met	ClinVar classifies this variant as Pathogenic, not Benign or Likely Benign. BP6 applies when a reputable source classifies the variant as benign. The ClinVar classification is Pathogenic, so BP6 is not met.	clinvar
BP7	N/A	BP7 applies to synonymous variants predicted to have no splice impact. This is a frameshift deletion, not a synonymous variant.

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